Revised Abstract: Inactivation of p53 is the most common genetic alteration detected in human cancers. Underscoring the importance of p53 in preventing tumorigenesis, mice in which p53 has been disrupted invariably develop lymphomas and other cancers. The overall goal of this proposal is to expand our understanding of the p53 tumor suppressor pathway, including upstream effectors and down-stream mediators, and the mechanisms by which it prevents pediatric cancers. Individuals diagnosed with Li-Fraumeni Syndrome (LFS) generally carry a germline p53 mutation and are remarkably predisposed to cancer at an early age. The mutations are highly penetrant and are associated with brain, breast, bone and adrenal cancers. We have recently identified a group of unrelated pediatric patients with adrenocortical carcinoma (ACC) in which 35 or 36 patients have a identical germline p53 mutation (R337H). Analysis of intragenic polymorphisms eliminate a founder effect. Tumors deleted the wild-type allele and express high levels of mutant p53 in the nucleus, indicating that p53 is functionally inactive in vivo. However, these patients are not from cancer-prone LFS families and over-expression of 337H in cell culture-based assays fail to detect a functional defect. Our hypothesis is that 337H is functionally inactive under physiologic conditions and that this mutation contributes to the development of ACC through a loss of p53-mediated cell cycle arrest and/or apoptotic activities. To test this hypothesis, we propose to address the following specific questions: 1) Does the 337 H mutation disrupt p53 biological activity under physiological conditions?; 2) Does the 337H mutant have altered biochemical and biophysical properties?; 3) Does the 337H mutation contribute to tumorigenesis? These studies should establish a new class of p53 mutation, a low penetrant allele, that would previously go undetected but nevertheless, significantly contributes to the development of human cancers.